N-Ethylmaleimide-modified subfragment-1 and heavy meromyosin inhibit reactivated contraction in motile models of retinal cones

The mechanism of contraction in motile models of teleost retinal cones has been examined by using N-ethylmaleimide (NEM)-modified myosin fragments (NEM-S-1 and NEM-heavy meromyosin [HMM]) to prevent access of native myosin to actin filaments during reactivation of contraction. In the diurnal light/dark cycle, retinal cones of green sunfish (Lepomis cyanellus) and bluegill (lepomis macrochirus) exhibit length changes of more than 90 mum. The motile myoid region of the cone contracts from 100 mum in the dark to 6 mum in the light. Motile models for cone contraction have been obtained by lysis of dark-adapted retinas with the non-ionic detergent, Brij-58. These cone motile models undergo Ca(++)-and ATP-dependent reactivated contraction, with morphology and rate comparable to those observed in vivo (Burnside, B.,B. Smith, M. Nagata, and K. Porrello, 1982, J. Cell Biol., 92:198-206). The cone myoids contain longitudinally oriented actin filaments which bind myosin subfragment-1 (S-1) to form characteristic “arrowhead” complexes which dissociate in the presence of MgATP (Burnside, B., 1978, J. Cell Biol., 78:227-246). Modification of S-1 or HMM with the sulfhydryl reagent, NEM, produces new species, NEM-S-1 or NEM-HMM, which still bind actin but which fail to detach in the presence of MgATP (Meeusen, R.L., and W.Z. Cande, 1979, J. Cell Biol., 82:57-65). We have used NEM-S-1 and NEM-HMM to test whether cone contraction depends on an actomyosin force- generating system. We find that reactivated contraction of cone models is inhibited by NEM-S-1 and NEM-HMM but not by the unmodified species, S-1 and HMM. Thus, reactivated cone contraction exhibits NEM-S-1 and NEM-HMM sensitivity as well as Ca(++)- and ATP- dependence. These observations are consistent with and actimyosin-mediated mechanism for force production during cone contraction.     

Interaction between actin and HMM

It is shown that the interaction between actin and HMM results in a rapid precipitation of acto-HMM gel upon addition of MgATP. This is a simple demonstration of the idea that the formation of myosin filaments is not essential for mechanochemical reaction (muscle contraction) to occur and that the soluble myosin heads are competent to interact with actin to produce mechanical effect. Our findings also strongly support earlier suggestion that each head of one HMM molecule is able to bind to a different actin filament.     

Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle

The effects of 2,3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20 degrees C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20 degrees C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.     

Fluorescence properties and contraction characteristics of ANM (N-(1-anilinonaphthyl-4)maleimide)-labeled rabbit psoas muscle fibers

Fluorescence spectra of ANM-labeled, glycerinated rabbit psoas muscle fibers were recorded in relaxed, contracted, and rigor states. SDS polyacrylamide gel electrophoresis of the ANM-labeled muscle fibers indicated that proteins labeled with ANM were myosin heavy chain, C protein, and actin. In a relaxed state in the presence of ATP, myosin heavy chain was mainly labeled. During the transition from rigor to the relaxed or contracted state, there was a blue shift (about 5 nm) of the ANM emission spectrum. Similar experiments with FAM (N-(3-fluoranthyl)-maleimide)-labeled muscle fibers showed that these fluorescence changes were not artifacts due to the movement of muscle fibers. The fibers labeled in the ATP relaxing solution showed a marked decrease in both isometric force and unloaded shortening velocity (Vo), while in the fibers labeled in the rigor solution isometric tension was not markedly suppressed, though Vo decreased to the same extent as in the fibers labeled in the ATP relaxing solution. Fluorescence spectra of ANM-labeled HMM in different states were also measured. A fluorescence enhancement and a blue shift (about 5 nm) of the emission maximum were observed in HMM + MgATP or HMM + MgATP + F-actin in comparison with HMM + F-actin. These results suggest that the fluorescence spectra of the ANM-labeled muscle fibers reflect their conformational changes between the rigor state (in the absence of MgATP) and the relaxed or contracted state (in the presence of MgATP).     

Effects of MgATP, MgADP, and Pi on actin movement by smooth muscle myosin.

To test the idea that the in vitro motility assay is a simplified model system for muscle contraction, the MgATP-dependent movement of actin filaments by thiophosphorylated smooth muscle myosin was characterized in the presence of the products MgADP and inorganic phosphate. The dependence of actin filament velocity on MgATP concentration was hyperbolic with a maximum velocity of 0.6 micron/s and an apparent Km = 40 microM (30 degrees C). MgADP competitively inhibited actin movement by MgATP with a Ki = 0.25 mM. Inorganic phosphate did not affect actin filament velocity in the presence of 1 mM MgATP, but competitively inhibited movement in the presence of 50 microM MgATP with a Ki = 9.5 mM. The effects of ADP and Pi on velocity agree with fiber mechanical studies, confirming that the motility assay is an excellent system to investigate the molecular mechanisms of force generation and shortening in smooth muscle. The rate at which rigor cross-bridges can be recruited to move actin filaments was observed by initiating cross-bridge cycling from rigor by flash photolysis of caged MgATP. Following the flash, which results in a rapid increase in MgATP concentration, actin filaments experienced a MgATP-dependent delay prior to achieving steady state velocity. The delay at low MgATP concentrations was interpreted as evidence that motion generating cross-bridges are slowed by a load due to a transiently high percentage of rigor cross-bridges immediately following MgATP release.     

Induction of contractile activity by rigor complexes in myofibrils

The relation between ATPase rate and substrate concentration was investigated for myofibrils with varying amounts of added HMM. There was a biphasic, 3 to 5-fold increase in ATPase in the absence of Ca⁺⁺. In the absence of added HMM, the peak activity occurred at ≤ 0.1 mM MgATP. With increasing concentrations of HMM, the position and magnitude of the ATPase peak shifted to larger substrate concentrations and higher rates. The cofactor activity of regulated actin in myofibrils is activated to a similar degree by Ca⁺⁺ as by HMM (rigor links). SDS gel electrophoretic patterns of myofibrils mixed with HMM indicated the soluble HMM binds to myofibrils at 0.1 mM MgATP and is dissociated at higher MgATP concentrations. Thus, in well-regulated myofibrils in the absence of Ca⁺⁺ actin cofactor activity can be activated by rigor complexes.     

Two pathways for oxygen exchange by heavy meromyosin and their dependence on actin

In the hydrolysis of MgATP by acto heavy meromyosin (HMM) there are two enzymatic pathways that differ in the properties of their intermediate oxygen exchange; one of these is designated the low exchange pathway (P1); the other is designated the high exchange pathway (P2). A plot of the P1 flux versus the actin concentration gives a sigmoid curve, whereas the corresponding curve for the P2 flux rises in an approximately hyperbolic manner. At low concentrations of actin, where the sigmoid curve of the P1 flux is in a lag phase, the major flux is along P2; but at higher concentrations of actin, as the P1 curve rises sharply, the flux along P1 comes to predominate. Even at the highest levels of actin, at saturating levels for both pathways, the kinetics of exchange along P1 and P2 are significantly different. In addition to these differences in the actin dependence, the flux of P1 relative to P2 is markedly inhibited by KCl. Therefore, which of the two pathways dominates during the hydrolysis of MgATP by HMM is strongly dependent on experimental conditions. The findings suggest that P1 involves the interaction of HMM with two actin units whereas P2 involves the interaction of HMM with one actin unit. The results are discussed in relation to a kinetic scheme based on this proposal.     

Calcium-sensitive binding of heavy meromyosin to regulated actin requires light chain 2 and the head-tail junction

Sedimentation in a preparative ultracentrifuge was used to determine the affinity of heavy meromyosin, HMM, for regulated actin, F-actin plus troponin-tropomyosin, in the presence of MgATP at pH 7.0, 20 degrees C, and mu = 18 mM. HMM was prepared from vertebrate striated muscle myosin by a mild chymotryptic digestion. This HMM contained 85-90% intact 19 000-dalton light chains, LC2. In the presence of calcium, 90% of the HMM bound to regulated actin with an association constant of (2-4) X 10(4) M-1. In the absence of calcium, while one-third of the HMM bound with an affinity similar to that observed in the presence of calcium, the rest bound much more weakly. It was not possible to accurately determine the association constant for this weakly binding HMM, but a 20-fold reduction in affinity is consistent with the binding data. The binding of single-headed heavy meromyosin to regulated actin was similarly sensitive to the calcium concentration. Since removal of calcium inhibits the regulated actin-activated ATPase of HMM greater than 20-fold, troponin-tropomyosin must be capable of inhibiting both the binding of HMM to regulated actin and a step which occurs after binding but prior to product release. Removal of LC2 increased the fraction of HMM with calcium-insensitive binding, and adding LC2 back to this depleted HMM restored most of the calcium sensitivity. Chymotryptic cleavage of LC2 to a 17 000-dalton fragment destroyed the calcium-sensitive binding of HMM to regulated actin. Phosphorylation of LC2, however, had no detectable effect on this binding. Thus, the calcium-sensitive binding of HMM to regulated actin requires that both the head-tail junction and the N-terminal part of LC2 be intact. Binding studies with cross-linked regulated actins and kinetic measurements of the rates of change in turbidity demonstrate that this calcium sensitivity is due to calcium binding to troponin and not to LC2.     

The binding of myosin heads on heavy meromyosin and assembled myosin to actin in the presence of nucleotides. Measurements by the proteolytic rates method

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin and myosin subfragment 1 were determined for the free proteins and their complexes with actin in the presence and absence of MgATP. The proteolytic reactions were carried out at 24 degrees C and under ionic strength conditions (mu) adjusted to 35, 60, and 130 mM. The percentages of myosin heads and myosin subfragment 1 bound to actin in the presence of MgATP were calculated from the rates of proteolysis for each set of digestion experiments. In all cases, the myosin heads in the synthetic filaments showed greater binding to actin than myosin subfragment 1. This binding difference was most prominent (3-fold) at mu = 130 mM. The binding of heavy meromyosin (HMM) to actin in the presence of MgADP was measured at 4 degrees C by ultracentrifugation and the proteolytic rates methods. Ultracentrifugation experiments determined the fraction of HMM molecules bound to actin in the presence of MgADP, whereas the proteolytic measurements yielded the information on the fraction of HMM heads bound to actin. Taken together, these measurements show that a significant fraction of HMM is bound to actin with only one head in the presence of MgADP under ionic conditions of 180 and 280 mM.     

Interaction of actin with myosin A and heavy meromyosin.

Ca2+ "free" actomyosin suspensions as well as actin heavy meromyosin (HMM) solutions in the presence of Ca2+ showed no contractile response (superprecipitation) and had low steady-state Mg2+-ATPase activity. Under the same experimental conditions both the enzymatic activity increased and contractile response was restored if the solubility of the proteins was depressed by the addition of polyethylene glycol 4000 (PEG-4000). The stability of the enzymatically active actomyosin or actin HMM complexes was 10 times lower in cleared solutions than in the insoluble actomyosin or actin HMM suspensions. It was concluded that soluble actomyosin or actin HMM solutions are inadequate test tube models for studying muscular contraction.     

Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.     

Evaluation of mechanical parameters of the mechanochemically 'active state' of actin filaments on the basis of purely chemical data

Oosawa and his collaborators (cf. F. Oosawa, Biophys. Chem. 11 (1980) 443), employing various optical techniques, have shown that the flexibility of actin filaments increases upon interacting with the enzymatically active myosin fragments, particularly heavy meromyosin (HMM). It has been reported (S. Hitchock, L. Carlsson and U. Lindberg, Cell 7 (1976) 53) that HMM can accelerate the DNase 1-induced depolymerization of F-actin, provided MgATP is also present. Since, as we have demonstrated (cf. J. Borejdo myosin, is endowed with mechanochemical capability, we made an attempt to correlate the enhanced rate of depolymerization with the decrease in rigidity of the G-G bonds in F-actin. On the basis of the chemical kinetic data of Hitchcock et al. we could derive the approximate value of the HMM-MgATP-induced change in rigidity which is a mechanical molecular parameter. Since interaction between HMM or HMM subfragment-1 and F-actin in the presence of MgATP leads to the movement of the myosin heads along the actin filaments, it is argued that the enzymic behavior of this system should not be analyzed on the basis of simple, equilibrium, complex formation.     

Muscle contraction and movement of cellular organelles: are there two different types of mechanisms for their generation?

It has been shown that myosin molecules attached to Covaspheres can "walk along" polar actin filament in vitro. The driving force for this movement seems to explain only about 1% of the isometric tension developed by a muscle fibre. Therefore, the driving force for the bead movement seems to be incompatible with that found in muscle, and the bead movement cannot be considered as a model for muscle contraction. The origin of the bead movement may be related to a "molecular jet" process, resulting from the rapid ejection of the MgATP splitting products. This "molecular jet" might also explain the movements of many cellular organelles.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate     

Translational motion of actin filaments in the presence of heavy meromyosin and MgATP as measured by Doppler broadening of laser light scattering

Intensity fluctuations of laser light scattering were utilized in order to follow enhancement of translational motion of the actin-heavy meromyosin (HMM) complex in extremely dilute solutions accompanied by the hydrolysis of MgATP. Such enhancement was anticipated on the basis of the idea that active streaming along actin filaments should be associated with their mechanochemical reactivity. Native tropomyosin was added in order to stabilize actin in its filamentous form, thus allowing the reduction of actin concentration below 50 micrograms/ml to enable free movement of neighboring filaments and yet give a reliable signal. Analysis of the data in terms of Doppler broadening led to an approximate evaluation of the average velocity of translation of the mobile filaments. This velocity was found to increase with increasing HMM concentration up to a maximum attained at a molar ratio HMM/actin of 1:2, and then decreased. Total intensity measurements indicate that the mobile scatterer is actually a complex of HMM with an isolated actin filament. HMM subfragment-1 was found to be ineffective. These results suggest that cooperation between the two myosin heads is necessary for efficient induction of active streaming along isolated actin filaments.     

Mechanism for the inhibition of acto-heavy meromyosin ATPase by the actin/calmodulin binding domain of caldesmon.

Caldesmon, an actin/calmodulin binding protein, inhibits acto-heavy meromyosin (HMM) ATPase, while it increases the binding of HMM to actin, presumably mediated through an interaction between the myosin subfragment 2 region of HMM and caldesmon, which is bound to actin. In order to study the mechanism for the inhibition of acto-HM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lacks the myosin binding portion. The 38-kDa fragment inhibits the actin-activated HMM ATPase to the same extent as does the intact caldesmon molecule. In the absence of tropomyosin, the 38-kDa fragment decreased the KATPase and Kbinding without any effect on the Vmax. However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3-fold decrease in the Vmax, in addition to lowering the KATPase and the Kbinding. The 38-kDa fragment-induced inhibition is partially reversed by calmodulin at a 10:1 molar ratio to caldesmon fragment; the reversal was more remarkable in 100 mM ionic strength at 37 degrees C than in 20 or 50 mM at 25 degrees C. Results from these experiments demonstrate that the 38-kDa domain of caldesmon fragment of myosin head to actin; however, when the actin filament contains bound tropomyosin, caldesmon fragment affects not only the binding of HMM to/actin but also the catalytic step in the ATPase cycle. The interaction between the 38-kDa domain of caldesmon and tropomyosin-actin is likely to play a role in the regulation of actomyosin ATPase and contraction in smooth muscle.     

Inhibition of actin-tropomyosin activation of myosin MgATPase activity by the smooth muscle regulatory protein caldesmon.

Caldesmon inhibition of actin-tropomyosin activation of myosin MgATPase activity was investigated. greater than 90% inhibition of ATPase activation correlated with 0.035-0.1 caldesmon bound per actin monomer over a wide range of conditions. Caldesmon inhibited sheep aorta actin-tropomyosin activation of skeletal muscle heavy meromyosin (HMM) by 85%, but had no effect on the binding affinity of HMM.ADP.Pi to actin. At ratios of 2 and 0.12 subfragment 1 (S1):1 actin, addition of caldesmon inhibited the ATPase activation by up to 95%, but did not alter the fraction of S1.ADP.Pi associated with actin-tropomyosin. We concluded that caldesmon inhibited actomyosin ATPase by slowing the rate-limiting step of the activation pathway. At concentrations comparable to the ATPase measurements, S1 displaced caldesmon from native thin filaments both in the absence (rigor) and the presence of MgATP. We therefore concluded that caldesmon could displace S1.ADP.Pi from actin-tropomyosin only under exceptional circumstances. An expressed mutant of caldesmon comprising just the C-terminal 99 amino acids bound actin 10 times weaker than whole caldesmon but otherwise inhibited actin-tropomyosin activation with the same potency and same mechanism as intact caldesmon. Thus, the entire inhibitory function of caldesmon resides in its extreme C terminus.     

The identification of F actin in the pollen tube and protoplast of Amaryllis belladonna

The production of protoplasts from the pollen of Amaryllis belladonna has facilitated a more direct investigation of the physiological and mechano-chemical basis of streaming. Cytoplasm is removed from an actively streaming protoplast or intact pollen tube and layered on a coated grid in a solution containing a low free calcium ion concentration. Under these conditions 6 nm thin filaments are observed. The thin filaments are morphologically identical with F actin and bind rabbit muscle HMM, forming characteristic arrowhead complexes that are dissociated by subsequent treatment with MgATP.     

Smooth muscle gelsolin and a Ca2+-sensitive contractile cell model

Smooth muscle gelsolin, termed smooth muscle 90-kDa protein in our previous paper (Kanno et al. FEBS Lett. 1985; 184:202-206), was purified from bovine aorta. Antibody prepared against smooth muscle gelsolin was used to detect the presence of gelsolin in human lung fibroblast MRC-5 cells permeabilized with Triton X-100 (MRC-5 cell models). These cells contracted in the presence of MgATP and Ca2+ in doses over 1 microM. Immunofluorescence microscopy using phalloidin and antigelsolin antibody showed that gelsolin was distributed along the stress fibers, except for a marginal bundle of cells, when MRC-5 cells were growth-arrested in serum-depleted medium. Making use of immunoblotting and indirect immunofluorescence techniques, we demonstrated that gelsolin is not retained in the MRC-5 cell models. We used purified smooth muscle gelsolin as a specific agent to sever the actin filaments. Preincubation of MRC-5 cell models with gelsolin led to a destruction of stress fibers, in a dose- and Ca2+ -dependent manner. The contractility was also lost, in the same manner described above, thereby indicating that a continuous distribution of actin filaments within the stress fibers is required for cell contraction. Treatment of MRC-5 cells with the Ca2+ ionophore A23187 induced an extracellular Ca2+ -dependent contraction but not a massive destruction of stress fibers, thereby indicating that most of the endogenous gelsolin was inactive under these conditions. Our interpretation of these results is that increases in cytoplasmic Ca2+ concentrations are sufficient for the contraction but may be too transient to activate endogenous gelsolin and thereby disrupt the stress fibers. Indeed, the inhibition of contraction of the MRC-5 cell, as induced by smooth muscle gelsolin, required preincubation in the presence of Ca2+, before the addition of MgATP. These results suggest that destruction of the stress fibers by endogenous gelsolin, which leads to inhibition of cell contraction, may occur if the cytoplasmic Ca2+ is maintained at high concentrations for a few minutes.     

Effect of Calcium Activated Factor on Properties of Actin and Myosin of Rabbit Skeletal Muscle

The effect of calcium activated factor (CAF) on enzymatic properties of actin and myosin was investigated. SDS polyacrylamide gel electrophoresis revealed that CAF did not degrade actin, but a slight degradation was found in myosin during CAF digestion, which might have been due to contaminated protease (s) in CAF preparation. No influence was found in EDTA ATPase of myosin and polymerization of G-actin during CAF digestion. However, heavy meromyosin (HMM) ATPase activating ability of actin was slightly decreased during CAF digestion. Although CAF digestion slightly decreased the biological activity of myofibrillar proteins, a single sarcomere prepared by CAF digestion is a useful model for studying muscle contraction because of its almost intact contractility.